Drug delivery strategy utilizing conjugation via reversible disulfide linkages: role and site of cellular reducing activities

Advanced Drug Delivery Reviews 55 (2003) 199–215 www.elsevier.com / locate / addr

Drug delivery strategy utilizing conjugation via reversible disulfide linkages: role and site of cellular reducing activities Go Saito a,c , Joel A. Swanson b , Kyung-Dall Lee a , * a

Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109 -1065, USA b Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109, USA c Pharmacokinetics and Drug Delivery Research Laboratories, Sankyo Co., Ltd, Tokyo, Japan Received 25 April 2002; accepted 13 July 2002

Abstract The first disulfide linkage-employing drug conjugate that exploits the reversible nature of this unique covalent bond was recently approved for human use. Increasing numbers of drug formulations that incorporate disulfide bonds have been reported, particularly in the next generation macromolecular pharmaceuticals. These are designed to exploit differences in the reduction potential at different locations within and upon cells. The recent characterization of a novel redox enzyme in endosomes and lysosomes adds more excitement to this approach. This review focuses on understanding where and how the disulfide bond in the bioconjugate is reduced upon contact with biological milieu, which affects delivery design and the interpretation of the delivery strategies.  2002 Elsevier Science B.V. All rights reserved. Keywords: Disulfide bond; Reduction; Bioconjugate; Macromolecular delivery; g-Interferon-inducible lysosomal thiol reductase (GILT); Protein disulfide isomerase (PDI); Glutathione (GSH)

Contents 1. Introduction ............................................................................................................................................................................ 1.1. Bioconjugation strategies for drug delivery and choice of linkage ........................................................................................ 1.2. Disulfide bond-based bioconjugation ................................................................................................................................. 2. Cellular redox enzymes and redox agents .................................................................................................................................. 2.1. Reducing cytosolic space and oxidizing ER space .............................................................................................................. 2.2. Disulfide reduction at cell surface and early endosomes ...................................................................................................... 2.3. Disulfide reduction in endosomes and lysosomes ................................................................................................................ 2.4. Characterization of redox enzyme in the endocytic pathway: GILT ...................................................................................... 3. Creating sulfhydryls via chemical and molecular approaches ...................................................................................................... 3.1. Generating sulfhydryls through chemical linkers ................................................................................................................ 3.2. Site-directed conjugation using endogenous sulfhydryls or mutagenetically inserted cysteines ............................................... 4. Disulfide linkage-employing drug delivery systems.................................................................................................................... *Corresponding author. Tel.: 1 1-734-647-4941; fax: 1 1-734-615-6162. E-mail address: [email protected] (K.-D. Lee). 0169-409X / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0169-409X( 02 )00179-5

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4.1. Macromolecular delivery systems via endocytosis .............................................................................................................. 4.1.1. Attachment of cellular targeting moiety through sulfhydryls ...................................................................................... 4.1.2. Conjugation of membrane-disrupting agents for enhanced cytosolic delivery .............................................................. 4.1.3. Bestowing stability and instability upon drug delivery systems via disulfides .............................................................. 4.2. Direct cytosolic delivery of macromolecules across the plasma membrane ........................................................................... 4.3. Controlled disulfide cleavage in the systemic circulation ..................................................................................................... 5. In vivo applications ................................................................................................................................................................. 5.1. Antibody-S-S-toxin .......................................................................................................................................................... 5.2. Antibody-S-S-drug ........................................................................................................................................................... 6. Conclusion.............................................................................................................................................................................. References ..................................................................................................................................................................................

1. Introduction

1.1. Bioconjugation strategies for drug delivery and choice of linkage

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aspects of this subject, we will briefly discuss the existing examples of disulfide linkage-employing drug delivery with the mechanism in mind.

1.2. Disulfide bond-based bioconjugation Attachment of cellular and subcellular targeting moiety, delivery-enhancing molecules, or functional entities to drugs or their delivery systems has become an essential and important approach in the field of modern drug delivery. Its need is more acute for the strategies required for macromolecular delivery, as unique problems arise as the molecular weight of the drugs increases, including changes in cytotoxicity, pharmacokinetics, dynamics and metabolism. For bioconjugates, the nature of the linker between the pharmacologic agent and the deliveryaugmenting moiety dictates the degree of successful delivery and its outcome. In this review, we have attempted to focus only on covalent linkages, rather than the less stable yet sometimes advantageous non-covalent linkages such as high affinity ligand– receptor interactions and electrostatic complexation. Among various covalent linkages, the primary focus of this review is the readily reversible yet relatively stable linkage of disulfide bonds. As there exist several reviews on this subject as well as comparisons among various covalent linkages utilized for drug delivery (for reviews on bioconjugate strategies see Refs. [1,2]), the attention here is directed to the mechanism of disulfide bond reduction after the drug delivery system contacts the biological milieu. The key subjects of this review are the questions of where and how the disulfide bond in the bioconjugate is reduced, which impacts the design strategy as well as interpretation of the experimental results. After reviewing the mechanistic

A disulfide bond (–S–S–) is a covalent linkage which arises as a result of the oxidation of two sulfhydryl (SH) groups of cysteines or other SHcontaining material. In bacterial and eukaryotic cells, they are often found in secretory proteins and exoplasmic domains of membrane proteins, which face a harsh extracellular environment. In eukaryotic cells, cysteines are correctly bridged in the endoplasmic reticulum (ER) via the disulfide bond, which functions primarily to fortify the protein tertiary structure. Two distinct characteristics that render this bond attractive in designing drug delivery systems are its reversibility and its relative stability in plasma. Covalently bonded disulfides can be formed spontaneously by autoxidation of sulfhydryls, primarily via oxidation upon exposure to air, which can reversibly be cleaved in the presence of reducing agents such as dithiothreitol (DTT) and b-mercaptoethanol. The presence of a high redox potential difference between the oxidizing extracellular space and the reducing intracellular space makes the disulfide bond intriguing as a potential delivery tool. Thus, the covalent linkage is dependent on the locale of the construct relative to the cellular compartments; a controlled cleavage and release of reduced components can occur upon cell entry. Indeed, a number of bacterial toxins, such as diphtheria and cholera toxins and plant toxins such as ricin, consist of two protein subunits linked via a disulfide bond. They take advantage of the reversible breakage of the

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disulfide bond during the process of translocation across cellular membranes into the cytosol of host cells [3]. Studies on toxin conjugates using disulfide bonds were published as early as the late 1970s [4–6], and ever since the disulfide-based bioconjugation approach has been a popular conjugation method applied in a variety of cellular drug delivery systems. Successful applications of thiol-based conjugation have obtained targeted delivery and enhanced cytosolic delivery, improved pharmacokinetics and increased stability. Moreover, such conjugates are being used more frequently in polypeptide or protein-based systems and in plasmid gene and oligonucleotide formulations, some of which are discussed later in this review. These macromolecular agents are membrane-impermeant, due to their large molecular size or polyanionic nature, and are typically endocytosed by cells. However, despite the substantial biological evidence for reductive cleavage of disulfide bonds occurring in the endocytosed substrates, the subcellular locations of these putative reduction sites and the mechanisms by which endocytosed macromolecules are reduced remain poorly understood [7]. This review focuses primarily on recent discoveries regarding the disulfide reduction in the endocytic pathway, and on the related disulfide conjugation-based delivery strategies for macromolecules. In particular, we concentrate on the redox enzymes and small redox buffers that reduce disulfides at cell surfaces, in various endocytic compartments, and in the cytosol.

2. Cellular redox enzymes and redox agents

2.1. Reducing cytosolic space and oxidizing ER space From the perspective of cellular drug delivery, access to the cytosolic space of eukaryotic cells is restricted primarily to hydrophobic small drugs, which have relatively high membrane partition coefficients and permeability constants, i.e. they diffuse passively across the lipid membrane. Macromolecular drugs have low diffusivity, and the plasma membrane is the primary barrier to entering the cytosolic space. Their uptake via permeation through

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membrane is very low, and instead they are generally endocytosed, carried to the lysosomes, and degraded. The lumen of endosomes and lysosomes are inside the cell, but still topologically extracellular. These spaces are connected to the lumenal spaces of the ER and the Golgi apparatus, but separated from the cytosolic space by membrane. The redox potential of the lumen of the endocytic compartments is much less well characterized than the reducing cytosolic space and the oxidizing ER [8], or even the mitochondrion [9,10], which will not be discussed in this review. We will start by reviewing how known redox potential differences inside the cell, between cytosol versus ER, are maintained. In general, disulfide bond reduction, oxidation and isomerization are mediated by small redox molecules alone, or with the help of redox proteins. Redox proteins typically require the co-presence of small redox molecules or other enzymes to regenerate and retain their activity. Both factors seem to play major roles in maintaining a high free SH level in the cytosolic space [11]. The thioredoxin family enzymes regulate these processes [12]. Members of this family include ubiquitous cytosolic enzymes such as thioredoxin and glutaredoxin [13], all of which share a common active site motif –Cys–Gly–His / Pro– Cys–, two cysteines flanking two amino acids [12]. Although these enzymes can catalyze reactions in both redox directions, the process in the cytosol is predominantly reducing. This is due to the abundant presence of reduced glutathione (GSH) and thioredoxin reductase, which regenerate and maintain the cysteine catalytic active sites in the reduced state [12,14]. Glutathione ( L-g-glutamyl-Lcysteinylglycine) is the most abundant non-protein thiol-source in mammalian cells, reaching millimolar concentrations [15], and thus is another important determining factor in controlling cytosolic redox potential. The redox state of glutathione in the cytosol is, in turn, biased to the reduced state by the ratio of GSH to GSSG, which is greater than 100 in most cells. This ratio is maintained catalytically GSSG → GSH by glutathione reductase and NADPH [15,16]. Consequently, the vast excess of reduced GSH over oxidized GSSG, and the thioredoxin enzymes that are kept in the reduced state by the GSH, maintain a high reducing potential in the cytosol. As a result, the environment in the cytosol,

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and the topologically connected nuclear space, prevents disulfide bond formation. The lumen of the ER is maintained in an oxidizing environment. This is the intracytoplasmic compartment for synthesizing and processing secretory and membrane proteins. In mammalian cells, disulfide bonds are confined to secretory proteins and to the exoplasmic domains of membrane proteins that are exposed to and processed in the oxidizing milieu of the ER. Hwang et al. [8] demonstrated that glutathione was also responsible for the redox potential in the ER. The GSH / GSSG ratio was estimated to be between 1 and 3, supporting a redox state | 100 times more oxidizing than that in the cytosol. Protein folding and formation of disulfides are thus favored in this oxidizing environment. Protein disulfide isomerase (PDI), an ER protein of the thioredoxin family, plays an important role in these activities. In summary, the redox potential of the cytosol and the ER are largely controlled by the redox status of glutathione, i.e. GSH / GSSG with thioredoxin family enzymes acting as catalytic effector of that redox potential (Fig. 1).

Fig. 1. Cellular redox enzymes and redox agents at different locations within a cell. Reduction of the disulfide bonds in endocytosed macromolecular conjugates occurs via surface-associated redox enzymes such as PDI and endosome / lysosome redox enzyme, GILT with its putative co-factor cysteine. Redox potentials of the reducing cytosolic space and the oxidizing ER space are regulated by the ratio of GSH and GSSG.

2.2. Disulfide reduction at cell surface and early endosomes In contrast to the cytosol, GSH concentrations in the extracellular space are much lower; concentrations in plasma typically being | 10 mM [17]. The oxidizing environment in the extracellular space generally favors the maintenance of disulfide bonds in cell surface proteins, as in the exoplasmic domains of membrane receptors, and in secreted proteins like insulin and immunoglobulins. Nevertheless, some cell surface-associated redox enzymes possess reduced thiols [18]. PDI is expressed at plasma membrane in addition to its major site of localization in ER [19]. It contains a KDEL sequence at the C terminus which facilitates its retention in the ER lumen, causing it to cycle between the cis-Golgi and the ER through its interaction with KDEL receptor [20]. Despite its KDEL sequence and ER retention mechanism, however, PDI is also found throughout the secretory pathway and at the plasma membrane. Although these distributions may reflect saturation of KDEL receptors [21], PDI secretion may also be a regulated event, or perhaps cell type-dependent, as not all KDEL-sequence containing proteins overflow from the ER to the cell surface [22]. Recent reports indicate that plasma membrane-associated PDI indeed plays important specific roles at the cell surface. Couet [23] has suggested that the thyrotropin (TSH) receptor in human thyroid cells undergoes partial shedding catalyzed by surface PDI; based on their observations that shedding can be inhibited by anti-PDI antibodies or by a membrane-impermeant sulfhydryl blocker, and can be increased by inhibiting endocytosis and recycling. Cell surface PDI has also been identified in human B lymphocytes [24], platelets [25], rat hepatocytes [26] and rat pancreatic cells [27]. Involvement of surface-associated PDI in the disulfide bond reduction of endocytosed material has been demonstrated [28]. In Chinese hamster ovary (CHO) cells, anti-PDI monoclonal antibodies and a PDI-inhibitor, bacitracin, partially inhibited cleavage of disulfide bonds in the membrane-adsorptive conjugate, [ 125 I]tyramine-S-S-poly( D-lysine). The role and involvement of plasma membrane PDI in the endocytic compartment were also demonstrated for

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diphtheria toxin, which consists of two domains, A and B chains, connected via a disulfide bond. Reduction of the disulfide bond in this toxin is a prerequisite for release of A chain from the endosome into the cytosol, which is required for the biological and pharmacological function of the toxin that leads to cytotoxicity [29]. Although other proposed mechanisms have been suggested for this reduction [30,31], cytotoxicity could be inhibited with anti-PDI monoclonal antibodies [28] and by membrane-impermeant sulfhydryl blockers such as DTNB and p-chloromercuriphenylsulfonic acid [32]. These studies indicated that disulfide bond reduction of endocytosed macromolecules begins at the cell surface, and may continue after macromolecules are internalized, together with surface enzymes, into early endosomes (Fig. 1). More recently described NADH-oxidase (NOX) is another cell surface-associated protein with disulfide–thiol interchange activity similar to PDI [33,34]. Interestingly, activity of this enzyme in normal cells is regulated by hormone or growth factors, but is constitutively activated in cancerous cells such as HeLa and hepatoma cells [35]. Thioredoxin, a small ubiquitous protein ( | 12 kDa) in the cytosol, was also localized to surfaces of human white blood cell lines such as human B and T lymphocytes, monocytes and granulocytes [36]. Other redox-active cell surface enzymes may exist in addition to these enzymes [22]. The role of these enzymes in disulfide reduction of endocytosed material has not been investigated.

2.3. Disulfide reduction in endosomes and lysosomes The significance of the cell surface enzyme-mediated reduction of endocytosed material remains unclear [37]. First, the functional activity of the thioredoxin family members is optimal at neutral pH [38,39]. Thus, upon internalization, the catalytic activity is most likely reduced rapidly as the pH in the endosome / lysosome compartments drops, favoring reduction on cell surface and in the earliest endocytic compartments. Also, optimal catalytic activity of these enzymes requires regeneration by other accessory molecules such as glutathione. This

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complicates efficient function at the cell surface. In fact, a number of studies indicate existence of additional reduction schemes in the endosome / lysosome compartments. Feener et al. [7] demonstrated that when CHO cells were incubated with disulfide linked conjugates, [ 125 I]tyramine-S-S-poly( D-lysine), reduction started immediately (which was shown later to be suppressible by anti-PDI antibody [28]) and continued for 6 h. Addition of the membraneimpermeant sulfhydryl inhibitor, DTNB, blocked the initial phase of disulfide cleavage, but after a | 30min lag-time, the intracellular phase of reduction resumed, indicating that the reduction process continued in endosomal / lysosomal pathways [7]. Shen et al. [40] assessed the significance of surface disulfide bond reduction using methotrexate transport-defective CHO cells and methotrexate-S-S-poly( D-lysine) conjugates. The conjugates were cytotoxic upon internalization and subsequent reduction, but not when pretreated with a reducing agent, b-mercaptoethanol. The authors interpreted this to indicate that reduction at cell surface was insignificant. Antigen presentation provides further evidence for disulfide bond reduction in the endocytic pathway [41,42]. In antigen presenting cells (APCs), endocytosed protein antigens are degraded into small peptides by proteases before binding to MHC class II molecules. During this process, denaturation and unfolding of the internalized proteins are facilitated by the acidic environment of these compartments [43]. Disulfide bonds, however, are not susceptible to the lysosomal proteolysis and remain chemically stable in the acidic environment; they must be cleaved by different process(es) [44,45]. In earlier studies using [ 125 I]tyrosine-S-S-[ 131 I]a 2 -macroglobulin and [ 125 I]tyrosine-S-S-[ 131 I]transferrin to investigate reduction after internalization by primary cultures of mouse peritoneal macrophages, Collins et al. [42] suggested that lysosomes are the primary site of disulfide reduction of antigens. The disulfide bonds in the transferrin conjugates, which recycled between cell surface and early endosomes, were not reduced, whereas those in the a 2 -macroglobulin conjugates were reduced only after they reached the lysosomes, 15–20 min after uptake. Lloyd [46] and Pisoni et al. [47] demonstrated that cysteine is actively transported from the cytosol to the lysosome lumen via a

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specific transporter in a normal human fibroblast. Gainey et al. [48] reported cysteine-specific transport in lysosomes of a murine macrophage hybridoma and in endosomes and lysosomes of a B cell hybridoma cell line. The existence of these active transport systems and accumulation of cysteine molecules in lysosomes and endosomes implied that cysteine-dependent disulfide reduction accompanies endosomal / lysosomal proteolysis, and may help to maintain the catalytic activity of the lysosomal cysteine proteases (e.g. cathepsins B, H, and L). Incidentally and perhaps relevant, the resulting thioloxidized cysteine dimer, cystine, was shown to be pumped out from the lysosome lumen back to the cytosol via a distinct transporter, where it may again be reduced to cysteine by glutathione [49]. Defects in this cystine transport system are associated with cystinosis [49].

2.4. Characterization of redox enzyme in the endocytic pathway: GILT Although cysteine was claimed to be the physiological reducing agent in lysosomes [42,46,48], the co-presence of redox enzymes with a small reducing agent such as cysteine or glutathione could facilitate the process more efficiently [50]. In addition, reduction is inefficient in acidic environments since it requires deprotonation of thiols [7]. The presence of such enzymes, therefore, had been inferred for a long time, yet only recently was the first of its kind identified and characterized [38]. Gamma-interferoninducible lysosomal thiol reductase (GILT) is a 30kDa soluble glycoprotein, which is expressed constitutively in endosomes / lysosomes of APCs in multiple species including mice and humans [51,52], and is inducible by interferon (IFN)-g in other cell types such as fibroblasts, endothelial cells and keratinocytes [53]. This is notably the first reducing enzyme identified primarily in the endocytic pathway. GILT is synthesized as a 35-kDa molecular weight precursor containing a mannose-6-phosphate signal sequence (for delivery to lysosomes). Electron microscopic studies revealed that the precursor GILT colocalized with early endosomes, while the mature GILT was only found in MHC class II-containing compartments (MIICs) [54], indicating that GILT is trafficked through the endocytic pathway [38]. GILT

expression also colocalized with MHC class II and the lysosomal marker Lamp-2 in mouse dendritic cells [55]. It was suggested that this enzyme is also involved in disulfide reduction of protein antigens in early endosomes as well, since the precursor GILT was catalytically active [51]. This thiol reductase possesses distinctive characteristics compared to other known thioredoxin members. Firstly, its catalytic active site, –Cys–X–X–Cys–, does not have the common motif of –Cys–Gly–His / Pro–Cys– shared by members of the thioredoxin family. Secondly, the optimal pH for thioredoxin family members is typically neutral [37–39], and GILT has the pH activity optimum of 4.0–5.5. For instance, PDI, a member of the thioredoxin family, contains the –Cys–Gly–His–Cys– sequence with the pKa of cysteine at 7.3 [39], which is significantly lower than the typical pKa of | 8.5 observed in most thiols in proteins or peptides. This low pKa favors PDI to be active at physiological pH in the ER, since the cysteines require a deprotonation step in the process of nucleophilic attack in order to form a covalent enzyme-S-S-substrate intermediate, followed by the adjacent second thiol attack, resulting in the reduced substrate [56]. The question of how GILT, despite having a thioredoxin-like structure, achieves an even lower optimal pH remains to be elucidated [51]. Thirdly, in vitro studies have shown that GILT requires the co-presence of a reducing agent, such as DTT or cysteine (but not glutathione) to regenerate and retain its activity. Arunachalam et al. [38] pointed out that cysteine may be one of its endogenous reducing buffers in vivo, since the presence of a high concentration of cysteine has been postulated in lysosomes and also in the endosomes of certain cell types [46–48] (Fig. 1). The importance of GILT in reducing disulfides in endocytosed material in vivo is indicated by studies of GILT-knockout mice [55]. In wild-type mice, GILT was markedly expressed in lymph nodes, spleen and lung, where APCs play major roles, and only expressed weakly in kidney, liver, and muscle [55]. Using a well-characterized egg lysozyme (HEL) as a model antigen, it was shown that the presentation of two of the four chosen HEL epitopes by spleen-derived APCs (e.g. B cells and macrophages) from GILT knockout mice was abrogated. These results, along with the information from the

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HEL epitope sequences, showed that the observed disruptions in stimulating their epitope-specific T cell clones were due to the lack of disulfide bond reduction within or near those HEL epitopes in the APCs of the GILT knockout mice. The existence of GILT explains, at least in part, the reduction process for endocytosed protein antigens in APCs (e.g. macrophages, B cells and dendritic cells). Of interest is the expression and location of similar, related redox proteins in endosomes / lysosomes of other cells. A study that may add some clues in this respect is that of a CHO fibroblast cell line. Merkel et al. [45] prepared two types of CHO cell transformants: cell line A, a CHO cell transfected with murine MHC class II gene, which convert this cell effectively to APC; and cell line B, a hybrid of cell line A fused with a murine L cell fibroblast. The CHO cell line A could elicit a CD4 1 T cell response only to the antigens lacking disulfide bonds, whereas the cell line B efficiently processed even the antigens containing disulfide bonds, suggesting that some cells have decreased endosomal / lysosomal reduction capacity which could be complemented genetically. Moreover, when cleavage of disulfides in [ 125 I]tyramine-S-S-poly( Dlysine) conjugates was compared, cell line A showed significantly less cleavage than cell line B. It is not known which gene from the L cell fibroblast provided the activity to CHO cells. Cell lineage-dependent schemes of disulfide reduction in the endocytic pathway can be further supported indirectly from the finding that a B cell hybridoma transported cysteine both into endosomes and lysosomes, while in a macrophage hybridoma the transport activity was limited to lysosomes [48]. In fibroblasts, endothelial cells and keratinocytes, the induction levels of GILT may vary depending on the cytokine levels such as IFN-g in the surrounding milieu [53]. These observations imply that cell type-dependent variations in the disulfide reduction mechanism of endocytosed macromolecules could derive from a number of factors, including expression levels of redox proteins such as GILT and surface PDI, location of amino acid transporters in vesicular compartments, kinetics of vesicular trafficking, concentrations of disulfidecontaining drugs at cell surface, position of disulfides in the conjugates, and perhaps differences between transformed cell lines and primary cells. This indi-

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cates that disulfide-based drug delivery systems require careful attention to the choice of target cell. The significance of this issue will be further discussed in the development of the recently FDA approved antibody-based cancer drug, Mylotarg  .

3. Creating sulfhydryls via chemical and molecular approaches

3.1. Generating sulfhydryls through chemical linkers To conjugate two molecules via a disulfide bond, thiol groups need to be introduced into both structures, unless endogenous thiols such as cysteines are already present. Different approaches are used to incorporate a thiol or cysteine moiety in a drug. For oligopeptides and oligonucleotides, it is easy to introduce a cysteine or to derivatize with a thiol group during chemical synthesis. It is more difficult for small drugs, however, since they rarely possess a thiol moiety for conjugation, and modification often diminishes efficacy. There are, however, some exceptions, such as maytansinoid [57] and CC-1065 thiol-derivatives [58] and calicheamicin [59] introduced in antibody-targeted drug delivery. Thiol(s) in a protein can be prepared in different ways. When free sulfhydryls are absent, they can be chemically generated; a popular approach is to modify primary amines found in surface exposed lysine residues or in the N-terminal residue with commercially available heterobifunctional linkers. These include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) with a thiol-reactive pyridyl-disulfide moiety, N-succinimidyl S-acethylthioacetate (SATA) that can be deprotected after reaction with hydroxylamine to give a free thiol, or 2-iminothiolane which can complement the cationic charge of a primary amine lost upon conjugation [60]. Activated pyridyl disulfides are convenient for creating a disulfide linkage between two molecules [61]. The reaction with a thiol becomes far more efficient with the leaving group, pyridine-2-thione, even in a pH-neutral buffer, and this can be followed spectrophotometrically. In fact, this popular chemical synthetic approach is utilized in most disulfide-based bioconjugate systems. Stability of disulfide bonds in vivo can be

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problematic especially when the half-life of a delivery vehicle in the systemic circulation is long. Sterically hindered pyridyl disulfide linkers are also available, such as 4-succinimydyl oxy-carbonyl-2pyridyldithio toluene (SMPT) and possess increased serum stability [62]. If reversibility is inessential in a delivery scheme, non-cleavable thio-ether bonds with increased stability in vivo are more often adopted. This can be accomplished simply by reacting sulfhydryls with sulfhydryl-reactive maleimide or haloacetyl derivatives instead of pyridyl disulfide derivatives. Chemical approaches also have drawbacks. Since most of the cross-linkers are based on conjugation to primary amine of lysine, a commonly found amino acid residue in a protein that exists at multiple sites, this method typically yields heterogeneity in the degree and position of substitution.

3.2. Site-directed conjugation using endogenous sulfhydryls or mutagenetically inserted cysteines Another approach is to utilize endogenous cysteines, if they exist in the protein structure. Since cysteines normally occur at low frequencies in proteins, site-specific end products can be expected. For instance, a group from Bristol-Myers Squibb developed doxorubicin (Dox)-antibody conjugates by selectively reducing the interchain disulfides, a total of four S–S bonds, in the hinge region and between the light and heavy chains. They showed that eight equivalent Dox derivatives could be consistently conjugated to various antibodies using this method, while SH groups chemically obtained from modifying primary amines with SPDP resulted in a rather inconsistent outcome [63]. The thio-ether bonded conjugates to the endogenous cysteines, in which Dox is cleaved from the antibody in the acid-labile acylhydrazone bond, are presently in human clinical trials [64]. The endogenous single cysteine in a sulfhydryl-activated bacterial cytolysin (G. Saito et al. 2002, Gene Therapy, in press) and cysteines in papain [65] have been demonstrated to be convenient sites for reversibly conjugating polycation or PEG via a disulfide bond. Even if cysteine is not readily available for conjugation, it can be incorporated into recombinantly expressed proteins by site-directed amino acid

substitution. This has been a powerful approach, especially when combined with thiol-specific bioconjugate techniques, now that genetically altered proteins have become easier to design and prepare. A cysteine can be inserted in appropriate positions of a protein molecule to achieve site specificity and optimal molecular orientation for recognition between ligands and receptors. Cochran et al. [66,67] recently investigated the importance of molecular distance and orientation in T cell recognition of MHC class II-antigen peptide complex conjugated to various maleimide linkers using the MHC molecules that contained a cysteine genetically inserted at different sites. The site-directed bioconjugation strategy can be found in numerous drug delivery systems, particularly in attaching engineered antibodies (e.g. Fab9, single chain Fv, etc.) to a delivery vehicle [68–70]. A group at Celltech Therapeutics demonstrated a site-specific pegylation (derivatization with polyethylene glycol, PEG) via a thio-ether bond to E. coli-expressed recombinant Fab9, possessing a free cysteine at the hinge region; this did not compromise binding activity and significantly improved pharmacokinetic profiles [71]. Pegylation to a genetically introduced cysteine in staphylokinase, a 136-amino acid profibrinolytic agent, via disulfide and thio-ether bonds was also shown to increase plasma circulation time by decreased hepatic clearance [72]. Since the approval of PEG-ADA (adenosine deaminase) in 1990, pegylation chemistry has developed based on covalent attachment via lysine residues, including recently approved pegylated IFNa for chronic hepatitis C viral infections [73,74]. This method, however, as already discussed, generally results in heterogeneous end products. Thus, the thiol-targeted, site-specific and controlled conjugation, which is essential for pharmaceutical manufacture and consistent therapeutic effect, may become the standard conjugation chemistry of the next generation pharmaceuticals.

4. Disulfide linkage-employing drug delivery systems In addition to the advantage of site-specific conjugation, the reversible nature of the disulfide bond is exploited in a number of ways for drug delivery. As

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described in the previous sections, disulfides can be reduced by different mechanisms in different environments. Depending on the locale of a drug conjugate at which the reduction and cleavage of disulfides is anticipated, the delivery strategies are divided into three groups. Some examples are briefly discussed below.

4.1. Macromolecular delivery systems via endocytosis The use of disulfide bonds for delivery of macromolecular drugs via endocytosis has been supported by studies in vitro and in vivo. The first antibodytargeted chemotherapy drug, discussed in detail in the in vivo application section, that utilizes disulfide bonds to release anti-cancer drugs upon cellular internalization, recently received FDA approval [75]. Although various endocytic compartments have been demonstrated to possess reducing activities, including cell surface, endosomes, lysosomes and the Golgi apparatus, for the majority of the delivery schemes reviewed below, it is mechanistically unclear which redox enzymes or agents are involved in that reduction process. We will start from some of the recent applications in nucleic acid delivery.

4.1.1. Attachment of cellular targeting moiety through sulfhydryls In non-viral gene delivery systems, disulfide-based conjugation techniques have aided development of formulations with high transfection potency. A key weakness of non-viral vectors is their low transfection efficiency compared to viral vectors. Disulfidebased conjugation methods have helped improve efficacy. First, conjugation of a targeting moiety is routinely used to enhance receptor-mediated cellular uptake. Wagner et al. [76,77] demonstrated increased expression of reporter genes after incorporating transferrin-S-S-polylysine conjugates to condense and complex with plasmid DNA. Erbacher et al. [78] used integrin-binding peptide (RGD)-S-S-PEI conjugates prepared from the cysteine-containing peptide and SPDP-modified PEI. Similarly, single chain Fv, possessing a genetically engineered cysteine at the C-terminus, was conjugated to SPDP-polylysine and used to enhance receptor-mediated gene delivery [70]. Attachment of a targeting moiety to other

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delivery schemes is also achieved with similar bioconjugation methods [79,80]. Cleavage of a targeting moiety from a delivery vehicle, however, is typically inessential, and more stable thio-ether bonds are still more often utilized. For example, for more recent in vivo studies, Wagner’s group has switched the conjugation method from a random SPDP modification of the targeting ligand to a more site-specific approach; attaching polyethylenimine (PEI) to the sodium periodate-treated carbohydrate moiety of transferrin to give a Schiff base, followed by reduction [81]. In addition to the controlled conjugation benefit, the conjugates are more stable in the systemic circulation [82].

4.1.2. Conjugation of membrane-disrupting agents for enhanced cytosolic delivery Secondly, bioconjugation schemes are used to incorporate membrane-disrupting agents that can breach the endocytic membrane barrier. Delivery of internalized cargo from the endocytic compartments into cytosolic space has used membrane-active fusogenic peptides [83]. Membrane active peptide-SS-polycation incorporated in the gene and oligonucleotide delivery systems was reported using the peptides derived from viruses such as influenza hemagglutinin subunit HA-2 [84,85], and other synthetic sequences such as amphipathic peptides including GALA and KALA [86]. A more recently characterized cationic peptide from bee sting venom, melittin, capable of both membrane-disruption and nuclear targeting [87], was incorporated into melittin-S-S-PEI / DNA complex and dioleoyl-S-S-melittin cationic lipid / DNA complex [88]. The endosomolytic activities of these membrane-active peptides are largely controlled by their concentrations and conformation changes in the acidic environment, which trigger membrane insertion and aggregation [89]. The importance of the disulfide bond cleavage and release of peptides in these conjugates upon cellular internalization, however, is not clear; as the disulfide bonded conjugates alone or the conjugates complexed to DNA are hemolytically active at low pH without the presence of reducing agents [84,87,88,90]. The peptides are also incorporated through more non-specific conjugation approaches such as biotin-streptavidin non-covalent linkage [90], and non-covalent ionic interactions between nega-

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tively charged peptides and positively charged DNA / polycation complexes [90,91]. We recently developed a unique gene delivery system that utilizes the endosomolytic mechanism of LLO, a pore-forming protein from intracellular bacterium, Listeria monocytogenes, to enhance the cytosolic delivery of plasmid DNA (G. Saito et al. 2002, Gene Therapy, in press). Our delivery system takes advantage of the reversible nature of disulfide bonds and is distinct from the fusogenic peptidebased delivery. The functional hemolytic activity of LLO, a member of sulfhydryl-activated hemolysins [92], is regulated by the redox state of its single cysteine residue within its conserved domain near the C-terminus [93,94]. Thus, to complex with DNA, a pyridyl-dithio derivative of protamine was reacted to the cysteine to prepare LLO with a disulfide link to protamine, LLO-S-S-protamine. We demonstrated that the LLO-S-S-protamine conjugate or its DNA complex completely lacks pore-forming activity of LLO, yet regains activity upon reduction. In a reducing environment, the disulfide bond is cleaved between the cysteine of LLO and the protamine, and LLO is released as its hemolytically active form. The 1:1 conjugation allowed us to estimate that only 40–160 LLO-S-S-protamine molecules in each protamine / DNA complex of | 100 nm were sufficient to produce significant enhancement in the reporter gene expression, in contrast to the requirement of relatively high peptide amounts in fusogenic peptide-based delivery. Reduced, active LLO administered along with protamine / DNA complex caused rapid toxicity due to plasma membrane damage, while the same dose of LLO-S-S-protamine produced high gene expression without any cytotoxicity. This indicates that reduction occurs after the complex is internalized, such that activated LLO molecules are released from the complex and form pores in the membranes of endocytic / lysosomal compartments.

4.1.3. Bestowing stability and instability upon drug delivery systems via disulfides Another application of disulfide bond usage for drug delivery exploits its relatively stable covalent bond to modulate the stability of formulations. An example in nature is in the structure of protamines, which condense and stabilize the chromosomal DNA

in mammalian sperm. Sperm protamines are highly cationic small proteins with over 50% arginine, which renders them capable of condensing DNA through ionic interaction. In mammals, protamines also possess six to nine conserved cysteine residues, which further compact and package DNA tightly by forming inter- and intra-molecular disulfide bridges during spermatogenesis [95]. Reduction by cytosolic glutathione has been implicated in the decondensation of sperm DNA after fertilization [96]. Disulfide bonds also appear to be important in assembly and stabilization of viral particles [97]. Similar strategies have been applied to non-viral vectors and shown to be effective in stabilizing aggregation-prone DNApolycation formulations. Blessing et al. [98] used a cysteine-containing cationic detergent to condense DNA, which quickly dimerized via autoxidation into stable 23 nm DNA particles. DNA / polycation complexes were also prepared by covalently cross-linking polyamines with disulfide bond-containing chemical linkers such as a bis-imidoester cross-linker (DTBP) [99–101], and using synthetic oligolysine peptides incorporated with varying numbers of cysteines [102] and thiolated PEG-block-polylysine [103]. Although cross-linking DNA polyplexes through disulfides improves stability, diminishes aggregation, and extends the half-life in the systemic circulation [100], the gene transfer efficiency decreases with increasing numbers of disulfide bonds [100,102]. Oupicky et al. [100] suggested that the diminished transfection efficiency was due to the decreased endosomal escape of the cross-linked complex. Perhaps too much stability via disulfides leads to inefficient reduction and decondensation of DNA complexes in the endosomal environment. Promising results in the in vivo application of disulfide bonds to liposomal delivery systems have been recently reported [104]. Pegylated liposomes with prolonged circulation lifetimes broadened the liposome-based drug delivery applications [105]. However, grafting PEG on the liposome surface decreases the effectiveness of ‘pH-sensitive liposomes’ in the endosome [106]. To solve this problem, Kirpotin et al. [107] synthesized a cleavable PEG-S-S-PE (phosphatidylethanolamine) lipid and demonstrated in vitro that the destabilizing property of pH-sensitive liposomes, consisting of PE and cholesteryl hemisuccinate (CHEMS), could be re-

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stored in the presence of DTT at pH 5.5 with cleavable disulfide-containing lipids. Ishida et al. [104] further extended its in vivo applicability by showing the improved therapeutic efficacy of PEGS-S-PE over PEG-PE as a stabilizer for delivery of doxorubicin-loaded, anti-CD19-targeted, pH-sensitive, PE / CHEMS liposomes. Because the drug was released swiftly from the vehicle, and its clearance was high due to the rapid cleavage of disulfides in blood, the authors suggested that more significant improvements may be obtained using more stabilized formulations in blood circulation.

4.2. Direct cytosolic delivery of macromolecules across the plasma membrane To overcome the plasma membrane barrier for delivering macromolecules into the cytosolic space of cells, physical methods such as microinjection, scrape-loading and electroporation have been often used in vitro. In contrast to these conventional techniques, there are unique biological approaches to delivery across plasma membranes, bypassing the endosomal pathway, that employ ‘cell-penetrating’ [108], ‘fusogenic’ [109], or ‘pore-forming’ [110] features of viruses and bacteria. Among these approaches, disulfide bond can be and has been used for conjugating cell-penetrating peptides (CPPs) [111] such as HIV tat protein-derived peptides and penetratin. Although the recombinant DNA approaches can effectively produce CPP-cargo fusion proteins in large quantities, their applications are limited to peptide or protein cargos. Chemical conjugation via disulfides, on the other hand, enables the delivery of non-protein macromolecules such as oligonucleotides [112,113], in addition to proteins and peptides [114]. Reduction of the disulfide bond in these conjugates takes place quickly once the constructs are delivered to the reducing milieu of the cytosol. Hallbrink et al. [115] compared the penetration kinetics of different CPPs using (fluorophore)cargo-S-S-CPP(quencher) probes. CPPs in the conjugate were labeled with a fluorescent quencher, 3-nitrotyrosine, and the cargo was labeled with a 2-amino benzoic acid fluorophore. The disulfide cleavage in the cytosol relieved the quenched fluorescence, which was monitored in real time as increased fluorescence intensity.

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4.3. Controlled disulfide cleavage in the systemic circulation Despite the oxidizing environment, reduction of disulfide bonds does occur in the systemic circulation, due to the presence of low concentrations of cysteine ( | 8 mM in humans) and glutathione ( | 2 mM in humans) [116,117]. This may inactivate the disulfide bond-based delivery systems with long blood-circulating times [62,104]. Zalipsky et al. [118] have taken advantage of this weakly reducing environment, and have developed a prodrug approach to conjugating PEG or lipids via a dithiobenzyl carbamate linkage [119] to an aminecontaining small drug [120] or protein [121]. The disulfide bonds are gradually reduced to release the drug or protein in circulation. The linker is designed such that disulfide bond cleavage triggers decomposition, resulting in release of the amino component of the conjugate in its original structure. Trimble et al. [122] investigated the release kinetics of a model peptide from cysteine mutants of hemoglobin-S-Speptide conjugates in the presence of low concentrations of glutathione. They showed that the peptide release rates can be controlled by varying the sites of attachment on the hemoglobin surface or by inserting aspartates in the peptide linker region. Charge repulsion between the nucleophilic glutathiolate anion and the aspartates was postulated to be responsible for the retarded release in the latter case. The concept is attractive, but in vivo studies have yet to be done.

5. In vivo applications

5.1. Antibody-S-S-toxin While many of the investigations reviewed thus far have been limited to demonstrating their feasibility in a cell culture model, in vivo studies of disulfide bond-based bioconjugates have centered around those of antibody-S-S-toxin, immunotoxins that target diseased cells [123]. These delivery schemes are based on the efficient internalization of antibodies before release of cytoxic components. Although the first-generation immunotoxins, murine monoclonal antibodies chemically coupled to subunits of ribosome-inactivating proteins, per-

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formed well in vitro, the in vivo studies were not as successful. This was due in part to the instability of disulfides in the blood circulation [62]. To improve the stability, sterically hindered linkers, such as SMTP inserted with a methyl group adjacent to the disulfide, have been employed. These immunotoxins with methyl-hindered linkers have produced significantly greater anti-tumor activity compared to those with unhindered linkers, in both preclinical and clinical studies of non-Hodgkin’s (B cell) lymphoma patients [62,124,125]. More recently, disulfide linkers with higher hindrance, including a geminal dimethyl group, two methyl groups at each disulfide end or a phenyl group, have been compared and tested for their relevance to immunotoxin delivery [126,127]. The anti-tumor efficacies of these immunotoxins have not yet been evaluated. However, in a mouse model, b-phase half-life of the conjugates increased in correlation with hindrance degree, which also correlated with disulfide bond stability against cysteine or glutathione. Importantly, the IC 50 for cytotoxicity was comparable among these toxin conjugates; intracellularly unbreakable thio-ether linked conjugates produced an inactive immunotoxin, strongly indicating the significance of disulfide bond cleavage in the endocytic pathway.

5.2. Antibody-S-S-drug A number of small anti-cancer drugs have been tested both in vitro and in vivo. Early antibody-drug conjugates contained clinically-used anti-cancer drugs such as methotrexate, doxorubicin and mitomycin C conjugated via either non-cleavable (e.g. amide and succinimide bonds) or cleavable linkage (e.g. acid-labile linkers such as hydrazone and cis-aconitic bonds, and lysosomally degradable peptide linkers and disulfide bonds) (for review, see Refs. [1,2]). The well-accepted linkers meet the essential criterion that they remain stable in the circulation for long periods, yet specifically break upon cellular internalization. These are sterically stabilized disulfide bonds (discussed below), acylhydrazone bonds [64,128,129] and degradable peptide linkers [130] (used in HPMA polymer drug conjugates). With the use of humanized antibodies and discovery of highly potent cytotoxic drugs, recent results of a so-called ‘magic bullet’ strategy in

human clinical studies have been encouraging. In fact, the first reversibly linked antibody-targeted drug, anti-CD33 antibody-S-S-calicheamicin, Mylotarg  , developed by Celltech Group and American Home Products for treatment of acute myeloid leukemia, won FDA approval recently [75]. To our best knowledge, this is the first drug on the market that contains a synthetic disulfide bond in its structure (the linker also contains cleavable acylhydrazone bond). Immunogen is also licensing to a number of biotechnology companies a technology based on a maytansinoid derivative, another potent anti-cancer drug that can be conjugated to antibodies via a disulfide. The disulfide bonds used in these conjugates are also stabilized with either a monomethyl or geminal dimethyl group attached to the adjacent carbon. It is strategically important to know which of these cleavable linkers perform best in vivo. Unfortunately, there are no publications that compare the same drug conjugates with different linkers. Cleavage of these linkers releases different drug components resulting in varying efficacy and metabolism. This makes direct comparisons difficult, as in the case of methotrexate-antibody conjugates containing a disulfide or degradable peptidic linker [131,132]. On the other hand, Hamann et al. [129,133] have recently reported important findings on the choice of linker used in calicheamicin-antibody conjugates. The antiCD33 antibody-conjugate optimized for targeting leukemic cells (Mylotarg  ) contains two cleavable sites in the linker, a disulfide bond and an acylhydrazone bond. The latter linkage chemically undergoes pH-dependent hydrolysis, which is favored in the acidic pH of endosome / lysosome compartments, a strategy also employed in the doxorubicin immunoconjugate [134]. Conjugates were prepared with a non-cleavable amide bond that replaced the hydrazone bond. In these, hydrolytic release of the drug via a hydrazone linkage was required to retain the conjugate’s high potency both in vitro, in vivo and ex vivo [129]. Interestingly, when the same drug-linker was conjugated to an anti-MUC1 antibody, which recognizes mucin epithelial antigen expressed in many solid tumors, the hydrolyzable bond conferred no advantage; the disulfide bond alone was sufficient to provide good results in all preclinical models. The authors pointed out that

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these differences are attributable to the physiology of the target cells rather than the properties of the conjugate itself, and suggested that different linkers should be tested and optimized for different target cells [129].

6. Conclusion It is exciting to see the increasing number of drug formulations that have incorporated disulfide bonds in various distinct ways. Many of these include the next generation macromolecular pharmaceuticals that are still under in vitro optimization. Yet there are examples that have successfully reached the market or are in clinical trials. One of the most popular approaches has been to exploit the cellular reducing potential in the endocytic pathway to trigger the cleavage of disulfide bonds. Its reduction mechanism, the essential part of the delivery strategy, is still unclear. On the other hand, the recent discovery of the novel endosome / lysosome redox enzyme found in professional antigen presenting cells adds more excitement to this approach. The fact that the expression level and perhaps the location of redox enzymes involved in the reduction of endocytosed macromolecules are cell-type dependent implies that the characterization of these enzymes on a molecular basis in each targeted cell may become an useful and integral part of this delivery design.

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